Substance-conjugated complement component C1q

ABSTRACT

A substance-conjugated complement component Clq is provided. A substance such as signal emitting substances or cell function regulating substances is conjugated via a sulfur atom to at least one site of the component. The site is not involved in binding immunoglobulins. A marker-labelled complement component Clq is used for measuring a complement-binding antibody, an antigen, a neutralizing antibody or a substance produced internally of and at the surface of a cell or a microorganism by measuring the marker.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part application of applicationSer. No. 779,671 filed Sept. 24, 1985, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention;

This invention relates to a substance-conjugated complement componentC1q and a process for preparing the same, and a method for measurement,using the substance-conjugated complement component C1q. Moreparticularly, it relates to a complement component C1q conjugated withvarious substances including markers and cell function regulatingsubstances and a process for preparing the same, and also to a processfor measurement, using such a substance-conjugated complement componentC1q.

2. Related Art Statement;

It has hitherto been known to utilize the complement fixation reactionfor the measurement or determination of antibodies in blood serum andantigens, such as microorganisms, phisiologically active substances andchemicals. This known method makes use of the serial reactions whereincomplement components C1 to C9 are bound successively to an antibodyspecifically bound to an antigen. In detail, this known method comprisesthe step of adding an excess amount of complement components to theformed antigen-antibody complex, the step of determining an amount ofresidual complement components through the hemolysis, and the step ofdetermining the amount of fixed complement components from the degree ofhemolysis. The quantity of the antigen or antibody is then estimatedfrom the results of the amount of the fixed complement components. Inthe hemolysis, complement components act on the sensitized erythrocytesincluding sheep red blood cells and anti-sheep red blood cell antiseraso that the complement components may be determined while using thehemolysis of the sheep red blood cells as the index. However, practicaldetermination operation of the hemolysis is extremely complex and needshigh level skill and knowledge. In addition, this known method has arelatively low sensitivity and requires two days for the determinationoperation.

Various methods have been proposed to overcome the aforementioneddisadvantages of the known method as described in the precedingparagraph. For example, Japanese Patent Laid-Open Publication No.43498/1980 discloses one of such methods. In the method proposed by theantecedent Publication referred to above, an antibody which binds, as anantigen, a complement component being bound to another antibody islabelled with an enzyme, and the amount of the thus labelled antibody isdetermined by the enzymatic activity thereof. This method is, therefore,one of the so-called enzyme-labelled antibody techniques. However, thismethod involves two step reactions, since a labelled antibody whichbinds, as an antigen, a complement component must be used. Accordingly,rinsing operations are required after each of the reactions, leading toincrease in labor and time. In fact, this determination method costsmuch time as several hours.

On the other hand, a method of determining a neutralizing antibody hasbeen made known, for example, by Takashi Kitamura, "Tissue CultureTechnology for Inspection of Virus", published by KINDAI SHUPPAN (1980),page 246. When an antibody against poliovirus, for instance, isdetermined by this method, cultivated cells originated from human being,a monkey or an ape are first inoculated with the poliovirus. (Meanwhile,the poliovirus does never grow if it is inoculated into cells originatedfrom the sources other than human being, a monkey or an ape.) The cellsinoculated with the poliovirus collapse and are deseased as the resultof cytopathogenesis due to propagation of the virus. However, thereaction product of a neutralizing antibody and the virus, (theinfectiousness of virus being neutralized by the neutralizing antibody),can not propagate even if it is inoculated upon a cell originated fromhuman being or monkey so that the cell is kept to have normal form andfunctions. Making use of this principle, a specific virus is reactedwith blood serum and then the titre of the neutralizing antibody isdetermined by inspecting the presence or absence, and the degree ifpresent, of plaque and CPE (cytopathogenic effect).

However, when the poliovirus is determined by the method described inthe preceding paragraph, the poliovirus must be cultivated for about 7days in a normal test in addition to the fact that the inspection andjudgement of the result should be made by a skilled person rather thanbeing easily conducted by a person having ordinary or middle levelskill. For this reason, an order of test for the determination ofneutralizing antibody is not accepted even by a large scale inspectioncenter at the present day.

On the other hand, as a method for determining antigens or antibodies ina simpler way, there has been known in the art a method whereinproperties of complement component C1q binding an antigen-antibodycomplex is utilized. (Simpson et al.,"Jounal of Immunological Methods",Vol. 67, 167 to 172 (1984).) In this known method, glutalaldehyde orperiodic acid is conjugated to the complement component C1q as across-linker, and peroxidase (enzyme) is conjugated via saidcross-linker to the complement component C1q as a marker. Marchalonis J.J., "Biochemical Journal", Vol. 113, pp229 to 305 (1969) discloses amethod in which radioactive iodine is conjugated to the complementcomponent C1q through the chloramine T method as a marker. However, inthese known methods, an enzyme or radioactive iodine is coupled witheach of the complement component C1q molecules via an amino grouppresent on the molecule generally and at random, resulting in entiremodification of the molecule since the very site of each molecule havinginherent properties capable of binding to an immunocomplex has beenchemically modified by said cross-linker or coupler. Accordingly, thebinding activity of such a marker-labelled complement component C1q forbinding to an antigen-antibody complex is seriously lowered to a levelnot to adapt for quantitative measurement as a reagent. Moreover, afalse-positive reaction takes place frequently by the latent presence ofsaid cross-linker in the marker-labelled complement component C1q tomake it impossible to continue the determination operations. It has,thus, been impossible to provide a reliable determination method fordetermining an antigen or antibody in a precise and reproducible mannerby the use of the complement component C1q.

OBJECTS AND SUMMARY OF THE INVENTION

It is, therefore, a primary object of this invention to provide acomplement component C1q which is conjugated with a variety ofsubstances, such as markers or cell function regulating substances,while preserving its inherent binding capacity for binding with animmunocomplex without any detraction, and a process for preparing such acomplement component C1q, and to provide a determination method in whichsuch a complement component C1q is used in a substance-conjugatedcondition.

Another object of this invention is to provide a complement componentC1q which is conjugated with a variety of substances to be convenientlyused as a determination or detection reagent for determining ordetecting a specific antigen present in a body fluid or held or bound toa cell or body tissue or for determining or detecting the correspondingantibody for the specific antigen, a modified immunoglobulin orimmunocomplex, and a process for preparing such a complement componentC1q, and to provide a determination method in which such a complementcomponent C1q is used in a substance-conjugated condition.

A further object of this invention is to provide a complement componentC1q which is conjugated with a variety of substances to be convenientlyused as a curing agent or medicine for regulating physiological functionof a variety of cells which have cell surface structures identified byspecific antibodies or which can capture specific immunocomplexes orcomplements, and a process for preparing such a complement componentC1q, and to provide a determination method in which such a complementcomponent C1q is used in a substance-conjugated condition.

A still further object of this invention is to provide a determinationmethod utilizing a complement component C1q, which is simple and easy indetermination operation and can be conducted for a short operation timeeven by an unskilled person.

Yet a further object of this invention is to provide a determinationmethod utilizing a complement component C1q, which has high sensitivityto give reproducible result of determination.

The above and other objects of this invention will be apparent from thefollowing detailed description thereof.

According to the present invention, there is provided a complementcomponent C1q wherein a substance is conjugated via a sulfur atom to atleast one site of said component, said site being not involved inbinding immunoglobulins.

Also provided in accordance with this invention is a process forpreparing a substance-conjugated complement component C1q, comprisingthe steps of:

(a) adding a reducing agent to a complement component C1q to cleave atleast one S--S bond present at a site not involved in bindingimmunoglobulins thereby to obtain a reduced complement component C1qhaving at least one exposed --SH group; and

(b) conjugating a substance to said complement component C1q via saidexposed --SH group.

Further provided in accordance with the invention is a method formeasurement by the use of a complement component C1q comprising reactinga maker-labelled complement component C1q with a material to bemeasured, said complement component C1q being conjugated with the markerat at least one site not involved in binding immunoglobulins, thereby toobtain a reaction material having said marker, and measuring saidmarker.

BRIEF DESCRIPTION OF THE DRAWING

The single FIGURE, FIG. 1, of the appended drawing is a graph showingthe change in neutralizing antibody titer of an anti-HSV positive humanserum in Example 15, one of the examples of the invention.

DESCRIPTION OF THE INVENTION

The present invention will now be decribed in detail.

In general, after an antigen is bound to an antibody, a complement isbound to the antibody already bound with the antigen to destruct theantigen. The complement includes complement components C1q, C1s, C1r,C2a, C2b, C3a, C3b, C4b, C5b, C6, C7, C8 and C9, and each one of thesecomplement components binds to a specific antibody pertinently dependingon the specific immunological reaction or allergy rection. Thesecomplement components bind in a fixed order such that the component C1qbinds to the antigen at a first place, followed by binding of C1s andC1r to C1q, and then the other complement components bind serially.

After eager investigations with the estimation that asubstance-conjugated complement component having a utility when used asa determination reagent or a curing agent might be prepared byconjugating a variety of substances to the complement component C1q,which is the component coupled with an antigen-antibody complex at thefirst place, such that the subsequent binding between theimmunoglobulins acting as an antibody and the component C1q is nothindered, we have succeeded to allow a variety of substances toconjugate at sites of the complement component C1q other than the sitesat which the immunoglobulins are to be bound.

In detail, we have given attention to the presence of nine S--S bondslocated at the sites of the polypeptide molecule of the complementcomponent C1q, the S--S bonds locating at the sites remote enough foraffecting the site having the binding capability for the immunoglobulinsand being formed by the fourth cysteine residues from the N-terminals ofthe A, B and C chains of the polypeptide constituting the complementcomponent C1q so that they are apt to be attaked by a reducing agent andapt to conjugate with a substance readily.

In view of the presence of such S--S bonds, we have contemplated tocleave these S--S bonds by the action of a reducing agent to expose atleat one S--H group in a step (a) of the process provided by the presentinvention.

The reducing agents which may be conveniently used in this step (a)include those used conventionally, the examples being sulfur-containingcompounds, such as mercaptoethylamine, dithiothreitol,2-mercaptoethanol, cysteine and glutathione.

The reducing step is carried out under the condition that the complementcomponent C1q is not modified. Preferably, reducing may be effected bydissolving the complement component C1q in a buffer solution in which itexists stably and then it is attacked by a reducing agent. Examples ofthe buffer solution used for this purpose include a tris buffered salinecontaining 10% of sucrose, 1 mol of sodium chloride and 5 mM (millimols)of sodium ethylenediamine tetra-acetate, and a phosphate buffered saline(PBS). The reducing reaction is carried out, generally, at about -2° C.to 45° C. for about 30 seconds to 24 hours, the reaction temperature andtime being changed depending on the specific reducing agent used.

It is desirous that the thus reduced complement component C1q be storedin a buffer solution to be used in the subsequent step (b) afterremoving the excess reducing agent by means of a conventional method,such as dialysis, salting-out process or gel filtration.

The complement component C1q utilized in the present invention is aglycoprotein contained in the blood serum of animal, and has nature forbinding firmly to the immunoglobulins when the immunoglobulins containedsimilarly in the blood serum and acting as an antibody reactspecifically with the corresponding antigen. The complement componentC1q used in the invention may be isolated from various aminals includingsheep, rabbit, guinea pig, cattle, horse, dog, mouse and human being,and the fraction enriched in the C1q component may be picked up inaccordance with a conventional purifying operation. (In this connection,reference should be made to "Operations in Immunological Experiment B",published by the Japanese Immunological Society, pp 1376 to 1380 (1974),if necessary.)

In the process of the invention, a variety of substances is conjugatedvia the exposed SH group of the reduced complement component C1q at thesubsequent step (b). The substances to be used in the step (b) and to beconjugated with the complement component C1q via the exposed SH groupinclude signal emitting substances, such as enzyme substrates, dyestuffsor pigments, magnetizable substances, donors or acceptors for electrontransference, radioactive materials, metal compounds and metalcompositions, which emit signals detectable by the sensory organs orexternal instruments, enzymes or coenzymes which may be modified to emitdetectable signals or agglutinating substances which get together,thereby to be readily detected; cell function regulating substances, forexample, certain enzymes which act on the counter-substances conjugatedto the complement component C1q to provide the latter with anyfunctions; and substances, such as high polymer materials, which captureor fix the counter-substances conjugated to the complement componentC1q.

More specifically, examples of the enzyme substrate areo-nitrophenyl-β-D-galactopyranoside and 3-hydroxysteroid; and dye stuffsor pigments include hemoglobin, the redox dyestuffs, such as methyleneBlue, and fluorescent dyestuffs, such as fluorescein isothiocyanate.Examples of the magnetizable substance are organic irons, such scarbonic iron, and microcapsules containing iron, and complexes of ironwith proteins may also be used. The donors and acceptors for electrontransference include a wide variety of substances which take part in theelectron transference, and chlorophyll which may be energized to takepart in the electron transference is included in this group of substanceand preferably used in the invention. Examples of the radioactivesubstances are ¹²⁴ I-labelled albumin, p-chloro(²⁰³ Hg)mercuribenzoicacid, N-ethyl(2,3-¹⁴ C)maleimide and iode(1-¹⁴ C)acetamide.

The metal compounds and compositions, other than the corbonic ironreferred to hereinabove, which may be used in the invention include goldcolloid and iron-containing microbeads.

Examples of the enzymes are peroxidase, alkaline phosphatase,galactosidase and alcohol dehydrogenase; wheras examples of thecoenzymes are nicotinamide adenine dinucleotide, nicotinamide adeninedinucleotide phosphate, flavin adenine dinucleotide and flavin adeninedinucleotide phosphate.

Example of the agglutinating substances are agglutinative fine particlessuch as latexes or microbeads; and other complement components C1q, i.e.complement components C1q to be conjugated with the exposed SH group ofthe reduced complement component C1q. These substances per se formagglutinate masses, which may be conjugated with the reduced complementcomponent C1q. The other examples of the agglutinating substancesinclude a combination of antibodies such as IgG or IgM and antigenscorresponding to the respective antibodies, a combination of lectinssuch as peanut lectin or wheat embryo lectin and materials having thecorresponding saccharide structures, a combination of biotin and avidinand a combination of protein and IgM. In a case where these combinationsare used, one material forming the combination is conjugated with thereduced complement component C1q and then the other material forming thecombination is added to form agglutinate masses. If the agglutinatingsubstances are used as a marker, the marker is easily detected byturbidity.

A variety of substances may be included in the cell function regulatingsubstance and conveniently used in the invention, examples being surfaceactive agents; antibiotics having activities to vital membranes, such asamphotericin B, and antibiotics affecting the metabolisms of cells, suchas actinomycin D; trace essential nutrients or growth factorsindispensable for the growth of cells, such as selenium compounds,insulin, transferring and epidermal growth factor; hormones such ascorticosteroid; factors, participating in the manifestation of cellfunction, such as macrophage activation factor; factors participating inthe cell division, such as B cell division factor; anticancer agents,scuh as mitomycin C; and toxins such as ricin of toxin of castor bean.

For instance, an enzyme may be utilized as the signal emitting substancewhile being conjugated to the complement component C1q to act as amarker so that it is used as a determination or detection reagent fordetermining or detecting a specific antigen present in a body fluid orheld by or adhering to a cell or body tissue, or for determining ordetecting the corresponding antibody to said antigen, modifiedimmunoglobulin or an immunocomplex.

When an antibiotic is used as the cell function regulating substancewhile being conjugated to the complement component C1q, the conjugatedproduct may be used as a curing agent for controlling the physiologicalfunction of a cell having a cell surface structure which can beidentified by a specific antibody or a cell capable of capturing animmunocomplex or a complement.

In the aforementioned step (b) of the process of the invention, asubstance having a group which can be conjugated to the exposed -SHgroup may be directly conjugated to the reduced complement componentC1q, or a substance may be conjugated indirectly to the complementcomponent C1q via a second substance having a coupling function, forexample, via one or more spacers or cross-linkers. Examples of thesubstance which may be directly conjugated include activated thiolSepharose (Trade Name of Pharmacia Fine Chemicals Co.) andp-chloromercuribenzoate. Any substance having a group capable ofconjugating to the exposed --SH group of the complement component C1q,such as maleimide residue or --SH group, and also having another groupcapable of coupling with the substance to be coupled indirectly with thecomplement component C1q may be used as the coupling agent for suchpurpose.

The coupling agent or the second substance having a coupling function,of course, varies depending on the substance to be coupled thereby. Asan illustrative example, when peroxidase extracted from horseradich iscoupled by the N-hydroxysuccineimide ester ofN-(4-carboxycyclohexylmethyl)maleimide, both reagents are dissolved in abuffer having a pH value of 6.5 to 7.5 and containing sodiumethylenediamine tetra-acetate to allow them to react with eath other at30° C. for an hour. The reaction conditions are selected inconsideration of the characteristics of a cross-linker used. Other thanthe N-hydroxysuccineimide ester ofN-(4-carboxycyclohexylmethyl)maleimide referred to above,N-hydroxysuccineimide esters of m-maleimide benzoic acid,N-(4-carboxyphenylmethyl)maleimide and maleimide acetic acid may be usedas the cross-linker. A peroxidase having therein a maleimide group isproduced. The thus produced peroxidase having a maleimide group is mixedwith the reduced complement component C1q, and maintained in a bufferheld at pH 5.5 to 6.5 and containing sodium ethylenediaminetetra-acetate at 4° C. for 2 hours, whereby a complement component C1qconjugated with peroxidase is obtained.

At the final step, the fraction of the complement component C1q labelledwith peroxidase, which serves as a marker, and having the activitiesoriginated both from the peroxidase and the complement component C1q maybe picked up through the gel filtration

As another illustrative example, if an antibody is employed as theagglutinating substance, an antibody having a maleimide group isobtained by the similar procedures under the similar conditions asexplained hereinabove with respect to peroxidase extracted fromhorseradish. When the antibody having the maleimide group is mixed andreacted with the reduced complement component C1q, a complementcomponent C1q conjugated with the antibody is obtained.

As a further illustrative example, if another complement component C1qis employed as the agglutinating substance, the complement component C1qis firstly reduced by the procedures described hereinabove with respectto the step (a). Then, the reduced complement component C1q is put in abuffer solution containing ethylenediamine tetre-acetate andN,N'-para-phenylene dimaleimide at pH 5.5 to 6.5 and is maintained for20 hours at 4° C., so that conjugated complement components C1q eachconjugated via S--S bond to form an agglutinate mass and havingmaleimide groups are obtained. When the conjugated complement componentsC1q are mixed and reacted with the reduced complement component C1q, acomplement component C1q further conjugated with complement componentsC1q is obtained.

It is preferred that the step (b) of the process of the invention becarried out in the presence of a buffer for both of the reducedcomplement component C1q and the substance to be conjugated thereto, andcarried out in the presence of a buffer for the coupler or cross-linkerin case where such a substance having the coupling or cross-linkingfunction is used.

In the substance-conjugated complement component C1q provided accordingto this invention, the substance is conjugated at a site or sites havingno binding capacity with the immunoglobulins, so that the bindingcapability to the immunoglobulins inherent to the complement componentC1q is kept intact without being hindered by the conjugating substance.

Since the substance-conjugated complement components C1q providedaccording to this invention are conjugated with various signal emittingsubstances and cell function regulating substances without blocking thesites at which the immunoglobulins is to be bound, they may be used forvarious applications including determination or measurement reagentsgiving the reproducible results or curing medicines in which theirinherent capacities for binding with the immunoglobulins are utilized.

The method for the measurement or determination, according to theinvention, utilizing the substance-conjugated complement component C1qwill now be described. The signal emitting substances, as describedhereinbefore, may be used in the measurement method according to theinvention. Such a signal emitting substance may be utilized as a marker.In detail, a substance-conjugated complement component C1q having amarker conjugated at a site that is not adapted to bind with theimmunoglobulins is prepared, and then the thus prepared marker-labelledcomplement component C1q is allowed to react with another material to bemeasured, whereby a reaction product conjugated with the marker isobtained, Subsequently, the labelled marker is qualitatively orquantitatively analysed to measure a variety of antigens, antibodies,neutralizing antibodies, substances produced or appearing in cells or onthe surfaces of cells or microorganisms. In this manner, the method ofthe invention may be applied for comprehensive uses, including variousclinical inspections and diagnoses of diseases.

One group or category of the substances which may be measured by themethod of the invention includes complement-binding antibodies. By thedetermination of certain complement-binding antibodies, various diseasesof wide-ranging hosts including not only human beings but also animalsand plants may be diagnosed and judgement may be made whether the hostsare infected or not, the diseases which may be determined by the methodof the invention including infectious diseases caused by microorganisms,such as bacterium, chlamydia and virus, tumor and autoimmune diseasessuch as systemic lupus erythematodus. Particularly in clinicalinspection of such an infectious disease and autoimmune disease, it is acommon practice to inspect whether or not a specific antibody uniquelyappearing with a certain disease is present in the blood serum. Variousmeasurement or inspection methods are known, and the complement-bindingreaction is involed in one of the important inspection methods.Notwithstanding that this complement-binding reaction has a utility whenutilized in a method of inspecting a certain specific antibody, thereaction has not been frequently uesd, irrespective of the importancethereof, since it involves cumbersome determination operations andrequires extremely high level skill.

Under these circumstances, a considerable contribution in industrial andmedical fields is made by the provision of a simple and speedy methodbased on this principle is developed to give a reproducible measurementor determination value at high sensitivity.

In determination of a complement-binding antibody in accordance with thepresent invention, an antigen is initially fixed to a solid phase orcarrier, and then the fixed antigen is allowed to react with an antibodyand with a complement component C1q combined with a marker, followed byremoval of unreacted materials and then the marker is quantitativelyanalysed. Utilizable antigens include viruses and bacteria, such asvaricella-zoster virus, measles virus, rubella virus, influenza virus,herpes simplex virus, hepatitis virus, mumps virus and mycoplasmaphneumonitis; physiologically active substances such as interferon; andantigens against autoantibodies such as DNA. Initially, such an antigenis fixed to a solid carrier to form a solid phase. Whereupon, theoperations, particularly rinsing operations, can be carried out easilyas compared with the prior art technology in which a lquid phase must behandled. Any carries may be used as far as the antigen absorbed therebyis not easily released from the solid phase, the examples beingsynthetic high polymers such as polyvinylchloride and polystyrene,natural high polymers such as filter paper, and cells and tissues. Morespecifically, a microtiter plate and polystyrene beads may be referredto as illustrative examples. The antigen may be fixed to the solid phaseby fixing the same on the surface of the solid carrier through physicalabsorption of chemical covalent bond and the like. In case where a cellor tissue is used as a solid carrier, the antigen may also be fixedthereto by infection.

Then, the fixed antigen is allowed to react with the specific orcorresponding antibody and a complement component C1q combined with amarker. The antibody is the objective substance which is to be measuredor inspected by the method of the invention, according to the aspect ofthe invention discussed just now, and the examples thereof are bodyfluids, such as blood serum, cerebrospinal fluid and saliva. The bloodserum is used most frequently, since it contains the largest quantitiesof antibodies. The antibodies determined or measured by the method ofthe invention must bind to the corresponding antigens uniquely orspecifically, and must be capable of binding to the complementcomponents. However, almost all of the antibodies produced in livingbodies satisfy the aforementioned conditions, and hence they may bemeasured by the method of the invention.

The reaction of the antigen fixed to the solid phase, the antibody andthe complement component C1q conjugated with a marker takes placespontaneously by simply mixing the aforementioned three reactantstogether to complete the reaction for forming a combined product. Whilethe reaction temperature and reaction time vary depending on thespecific antigen or other reactants used, these conditions may beselected properly unless bioactivities are lost.

Since the complex composed of the antigen, the antibody and themarker-labelled complement component C1q is fixed to a solid phase, theunabsorbed complement component C1q and inhibitors for the reaction canbe readily removed by rinsing. The marker incorporated in the complex isthen quantitatively analysed. For quantitative analysis, any of theknown methods may be used, including visual measurement, observationsthrough various types of microscopes, measurement of absorbance ofvisual and ultraviolet rays, fluorophotometric measurement and pulsecount measurement. In determination of the marker, other than the directmeasurement of the marker labelled to the combined complex, a knownquantity of the marker is used and the quantity of the marker which hasnot been conjugated to the complement component C1q may be determined tolearn indirectly the quantity thereof introduced into the complex.Anyway, by the quantitative analysis of the marker, the quantity of thecomplement-binding antibody coupled to a specific antigen can belearned.

In the measurement or determination method of the invention, enzymes areparticularly preferred as the signal emitting substance. Since enzymesact as catalysts, the sensitivities of the measurement may be freelyadjusted by changing the temperature and time of the reaction.

Various antigens may also been inspected by the measurement method ofthe invention. The measurement of a variety of antigens may be utilizedfor the detection, identification, quantitative analysis and inspectionof various substances including microorganisms such as chlamydia andvirus, various physiologically active substances such as interferon andlymphokines, specific antigens for cancers, specific antigen substancesin immunological abnormalities, allergens in allergic diseases, andmedicinal substances such as hormones; and thus the method may beapplied for various purposes, such as speedy and reliable diagnosis on avariety of diseases, provision of the standards for judging the effectsof curing actions, inspection for doping, inspection for thedetermination of foreign matters in products, and hygienic or sanitaryinspections.

When a specific antigen is measured by the method of the invention, asubstance having affinity with the antigen which substance is fixed to asolid carrier is reacted with the antigen, and the complement componentC1q labelled with a marker, and optinally with an antibody, followed byremoval of unreacted materials and then the marker is quantitativelyanalyesd.

The substances having affinity with the antigen include various types ofsubstances by which the antigens are readily absorbed, examples thereofbeing antibodies, portions of antibodies containing the sites binding tothe antigens [for example, Fab, F(ab') or F(ab')_(z) ], enzymesubstrates and inhibitors, protein A of staphylococcus, variousmedicinal substances originated from organisms, and receptors for virus.A living tissue containing a substance having affinity with a specificantigen may be used without being purified. A selected one of thesesubstances having affinities with antigens is fixed to the solid phaseor carrier. By the use of the fixed phase, rinsing and other operationscan be simplified. Any carriers may be used for this purpose, as far asthe substances having affinities with antigens are not readily releasedor removed, and the same carrier materials as has been described for themethod of measuring the complement-binding antibodies may be used.

A specific antigen to be measured is then added to the fixed substancehaving affinity with the antigen so that the antigen is conjugated withthe substance. Any antigens may be measured by the method of theinvention without particular limitation, as fas as they can be coupledwith substances having affinities thereto, such as the correspondingantibodies. Illustative antigens which may be measured by the method ofthe invention include microorganisms such as viruses and bacteria,products produced by viruses and bacteria, vital components in animaltissues, physiologically active substances of plants, and chemicals.Sources for such antigens are body fluids such as blood, urine,cerebrospinal fluid and saliva, processed products of meats and plants,and aqueous solutions from rivers, sewege or waste water.

In the method of measuring an antigen, according to the invention, thecorresponding antibody is added optionally as necessity arises. When anantibody or a portion of antibody having an antibody-binding activityand a complement-binding activity is used as the substance having theaffinity with the antigen, it is not requisite to further add theantibody. However, in case where a susbstance having nocomplement-vinding activity is used as the substance having the affinitywith the antigen, it is essential to add the antibody to be bound to themarker-labelled complement component C1q. The antibody may be added atany desired time point after the antigen is added, and may be addedsimultaneously with the addition of the complement component C1q or maybe added before or after the addition of the complement component C1q.Of course, the antibody should be the one which binds uniquely to thespecific antigen to be measured and should be capable of binding withthe complement component C1q. Typical antibodies used commonly areimmunoglobulins contained in animal blood sera, the examples being IgM,IgG etc. having complement-binding activities. Natural antibodiespresent in blood sera may be used, or desired antibodies may be obtainedby administrating or infecting animals with antigens. In addition to theimmunoglobulins purified and separated from blood sera, inactivatedblood sera may be used as the antibodies without purification.

The condition for the reaction between the antigen, the substance havingaffinity with the antigen, and the marker-labelled complement componentC1q and the antibody if it is added is not ristricted. Only by mixingthe materials, the reaction proceeds spontaneously and quantitatively.While the time and temperature of the reaction vary depending on thespecific kinds of the antigen and the other reactants, the reactioncondition may be set with the only limitation that the biologicalactivities of the reactants are preserved.

Since the comlex of the substance having affinity with the antigen, theantigen and the marker-labelled complement component C1q is fixed to thesolid phase, the unreacted complement component C1q and inhibitors forthe reaction may be easily removed by simple rinsing operation. Themarker of the complex fixed to the solid phase is then quantitativelyanalysed. For the quantitative analysis, similar methods as has beendescribed in determination of complement-binding antibody may beemployed.

While in the above-mentioned methods for measurement a solid phase orcarrier is employed, it is possible to measure without the use of such asolid phase or carrier. Specifically, after an antigen, an antibody anda marker-labelled complement component C1q are reacted, the marker maybe measured directly without removing unreacted materials. In such acase, it is necessary to select a marker which is highly distinctive andis readily detectable. For example, if an enzyme such as peroxidase isused as a marker, the activity or sensitivity of the enzyme can beenhanced as the concentration of substrates is changed. Therefore, it ispreferred to use enzyme as a marker. As another method in which a solidphase or carrier is not used, after an antigen, an antibody and a markerlabelled-complement component C1q are reacted, a precipitating agent isadded to precipitate unreacted materials such as unreacted complementcomponent C1q and other unnecessary proteins followed by measuting themarker. The examples of the precipitating agent include materials whichlower the solubility of proteins or agglutinate masses thereof, such aspolyethyleneglycol, organic solvents such as ethanol, inorganic saltssuch as ammonium sulfate or sodium sulfate and organic or inorganicacids which change pH values of solutions such as trichloroacetic acidor hydrochloric acid. Basic materials may also be used to change the pHvalue. Ethylenediamine tetra-acetic acid or ethyleneglycol tetra-aceticacid may also be used as the precipitating agent since there compoundscapture metal ions in the solutions, thereby lowering the solubility ofproteins or agglutinate masses thereof.

Neutralizing antibodies may also be measured by the method of theinvention. Neutralizing antibodies are antibodies for preventinginfections by microorganisms, such as virus, rickettsia and chlamydia.Referring to diseases caused by virus, for instance, a wide variety ofviral infectious diseases have been known up to date, including not afew serious diseases. For instance, if a pregnant woman is infected withrubella virus, there arises a danger that a malformed baby is born.Fatal damages are caused by the infection with rabies virus, Japaneseencephalitis virus and poliovirus, with the nerval cells sufferedunrecoverable disorders, leading to lasting troubles throughout thelifetime. Hepatitis caused by hepatitis virus is an infectious diseasewhich lasts as a chronic disease for a very long time, and a portion ofthe liver is impaired by liver cirrhosis which might lead to hepatoma.

However, it is extremely hard to inhibit the growth of virus by the useof a variety of medicines including antibiotics, since a virus can growin special living cells, i.e. the susceptible cells, unlike bacteriumand fungi.

Accordingly, it is a more important counter-measure against the diseasescaused by virus to prevent infection by virus or to protect a personfrom infection, apart from the curing treatement of the patients. Thejudgement on the question whether a person is susceptible to infectionby a specific virus or not may be rendered by the determination ofpresence or absence of the neutralizing antibody to the virus underquestion and by the measurement of the titer of the existingneutralizing antibody.

A living body acquires sound immunity after it has been infected with aspecific virus and then recovered from the disease caused thereby. Thismeans, in fact, that a system for preventing the living body fromre-infection with that virus has been established. In other words, aliving body has been once infected with a specific virus, the antibodyfor protecting the body from re-infection is promoted, the antibodybeing referred to as infection preventing antibody. Production andpreservation of the infection preventing antibody are very importantfactors against the infection by the virus. After being infected orimmunized with a virus against the attack by the virus (for exampleinoculated by vaccine), various antibodies against the structuralcomponents of the virus have been produced in a living body. However,all of these antibodies produced in the living body are not participatedin the prevention against infection by that virus. Only the antibodyhaving the function for inhibiting the growth or propagation of thevirus is referred to as neutralizing antibody or infection preventingantibody. This particular neutralizing antibody exerts the principalrole in prevention of infection. The present invention provides a methodof measuring a variety of neutralizing antibodies against all viruses,rickettsias and chlamydias which infect culture cells speedily andquantitatively on a number of samples.

In the method of measuring a neutralizing antibody, according to theinvention, a liquid containing a known quantity of microorganism, suchas virus, rickettsia or chlamydia, is reacted with a body fluid to bemeasured, such as blood, cerebrospinal fluid, saliva or blood serum. Atthis reaction step, the neutralizing antiboy, if present, reacts withthe mixed microorganism. The amount of microorganism reacting with theneutralizing antibody is increased as the amount of neutralizingantibody contained in the liquid under measurement increases so that theamount of the residual microorganism is decreased. Since the content ofmicroorganism in the measured liquid is known, the amount ofneutralizing antibody contained in the measured liquid can be calculatedfrom the result of determination of the residual microorganism.

After the preceding reaction step, the residual microorganism isinoculated on culture host cells to allow to grow. The cultivation isstopped after the lapse of pre-set time, whereby fixed cells containingtherein the residual microorganism are obtained. A marker-labelledcomplement component C1q and an antibody against the microorganism arethen added to react with the fixed cell to obtain a modified fixed cellto which the marker-labelled complement component C1q and the antibodyare bound. By the quantitative analysis of the marker, the amount ofresidual microorganism can be learned to find the amount or titer of theneutralizing antibody.

The residual microorganism is, in general, cultivated initially byinoculating the microorganism on host cells cultivated through amonolayer culture on a micro plate to allow the microorganism to beabsorbed by the micro plate, and then allowing it to grow or propagateon the plate. It is a common practice to inactivate endogenous enzymesand the microorganism by treating with, for example, methanol-containinghydrogen peroxide. Antibodies against the residual microorganism whichmay be used in the method include antisera, such as low titer human seraand animal immunoe sera, and monoclonal antibody.

Furthermore, the materials which may be measured by the method of theinvention, other than those described above, are products produced orappearing internally of or on the surfaces of cells, and variousmicroorganisms. The materials belonging to this category include cellsurface antigens produced by cells, such as asialo GM₁, T antigen and Lyantigen; intracellular enzymes, such as TdT (terminal deoxynucleotidyltransferase), GTP (γ-glutamyl transpeptidase) and LDH (lactatedehydrogenase); secreting substances, such as CEA (carcino embryonicantigen) and AFP (α-fetoprotein) and immunoglobulins: and enzymes andpeptide base substances produced by yeasts and bacteria. The cellsreferred to above include all kinds of cells including animal cells,plant cells, heterokaryotes, cells of yeasts, bacteria and protozoa, andcells subjected to gene engineering. Microorganisms which may bemeasured by the method of the invention include all microorganismsinfecting cultivated cells, such as viruses, rickettsias and chlamydias.

In measurement, one of the aforementioned substances or microorganismsis cultivated and fixed, or simply fixed, and then reacted with acomplement-binding antibody and a marker-labelled complement componentC1q, followed by determination of the marker, whereby the substance ormicroorganism may be quantitatively analysed.

EXAMPLES OF THE INVENTION

The invention will now be illustratively described with reference toexamples thereof and comparative examples.

EXAMPLE 1 Enzyme-Conjugated Complement Component C1q:

(1) Purification

100 ml of a fresh rabbit blood serum was dialized through 5 l of a0.026M aqueous solution of ethylene glycol tetra-acetate (pH 7.5) for 15to 24 hours, and the formed precipitate was recovered by centrifugalseparation (20,000G, 20 minutes). The recovered precipitate wasdissolved in 20 ml of a 0.75M aqueous solution of sodium chloride (pH5.0) containing 0.1M of sodium ethylenediamine tetra-acetate. Afterremoving the insoluble materials by centrifugal separation (25,000G, 30minutes), and then the solution was dialized through 5 l of 0.063Maqueous solution of sodium ethylenediamine tetra-acetate (pH 5.0) at 5°C. for 4 hours, followed by removal of precipitates by centrifugalseparation (20,000G, 20 minutes). About 3 mg of proteins were obtainedby the aforementioned operations, and 95% or more of the thus obtainedproteins was occupied by the complement component C1q. In order to storethe complement component C1q, the proteins were dissolved in an aqueoussolution (pH 7.4) containing 0.05M tri(hydroxymethyl)aminomethane, 1Msodium chloride, 0.005M sodium ethylenediamine tetra-acetate and 10%sucrose. The aforementioned operations sequence may be repeated tofurther purify the complement component C1q.

(2) Preparation

30 mg of the thus purified rabbit complement component C1q was dissolvedin 10 ml of an aqueous solution (pH 7.4) containing 0.05Mtris(hydroxymethyl)aminomethane, 1M sodium chloride, 0.005M sodiumethylenediamine tetra-acetate and 10% sucrose. The solution was thenadded with 0.1 ml of a 0.1M dithiothreitol, and allowed to stand at roomtemperature for an hour for reaction. The reaction solution was thenpassed through a Sephadex G-25 (Trade Name of Pharmacia Fine ChemicalsCo.) column to recover the protein fraction which was concentrated tohave a volume of about 10 ml by ultrafiltration to obtain 22 mg of areduced complement component C1q.

Separately, 20 mg of peroxidase extracted from the horseradish wasdissolved in 6 ml of a phosphate buffer (pH 7.4), and then added with 4ml of a dimethylformamide. The solution was further added with 0.2 ml ofa 2% 4-(maleimidemethyl)-cyclohexane-1-carboxylic acid succineimideester (hereinafter referred to as CHM) in dimethylformamide, and thenallowed to stand at room temperature for an hour for reaction. After anhour, the solution containing the reaction product was passed through aSephadex G-25 column to recover 16 mg of a CHM-conjugated peroxidase.

21 mg of the aforementioned reduced complement component C1q and 14 mgof the CHM-conjugated peroxidase were mixed together, and the mixturewas allowed to stand stationarily at 4° C. to 10 ° C. for 15 hours andthen passed through a Sepharose 6B (Trade Name of Pharmacia FineChemicals Co.) column to recover a fraction of a molecular weight rangeof from 400,000 to 800,000 to obtain 29 mg of a complement component C1qlabelled with peroxidase.

EXAMPLE 2 Enzyme-conjugated Complement Component C1q

1 mg of β-D-galactosidase derived from Escherichia coli was dissolved in0.2 ml of a 0.1M phosphate buffer (pH 6.0), and then reacted with 0.1 mgof N,N'-o-phenylenedimaleimide dissolved in 0.2ml of a phosphate buffercontaining 5% dimethylformamide at 30° C. for 25 minutes. The solutioncontaining the reaction product was passed through a Sephadex G-25(Trade Name of Pharmacia Fine Chemicals Co.) column equilibrated with aphosphate buffer containing 0.2 mg/ml of bovine serum albumins, wherebya 720 μg of β-D-galactosidase coupled with maleimide.

200 μg of the β-D-galactosidase coupled with maleimide was dissolved in0.1 ml of a phosphate buffer containing 1 mM of sodium ethylenediaminetetra-acetate, and reacted with 2 mg of the recuced complement componentC1q prepared in Example 1 and dissolved in 0.1 ml of a phosphate buffercontaining 1 mM of sodium ethylenediamine tetra-acetate at 4° C. for48hours. The solution containing the reaction product was subjected togel filtration using a Sepharose 6B (Trade Name of Pharmacia FineChemicals Co.) column, and then processed through the procedures asdescribed in Example 1 to obtain an active fraction, i.e. a fractioncontaining 1.6 mg of a complement component C1q combined withβ-D-galactosidase.

EXAMPLE 3 Enzyme-conjugated Complement Component C1q

(1) Preparation

Generally following to the same procedures as in Example 1, except thata goat serum was used in place of the rabbit serum, a reduced complementcomponent C1q was prepared, which was then conjugated with peroxidase toobtain a peroxidase-labelled complement component C1q.

(2) Test

Using a serum having a CF antibody titer of 16, the reactions of thethus obtained peroxidase-labelled complement component C1q with a herpessimplex virus CF antigen and with a normal cell antigen were inspectedby means of the solid phase enzyme immunoassay to obtain the results asset forth in the following Table 1. In Table 1, the results of thisExample are shown together with the results of the following ComparativeExample 1.

COMPARATIVE EXAMPLE 1 Enzyme-conjugated Complement Component C1qPrepared by Conventional Process and Having the Enzyme ConjugatedGenerally at Random

(1) Preparation

1.5 mg of horseradish peroxidase was dissolved in 0.2 ml of distilledwater, and added with 60 μl of a 0.1M sodium periodate solution,followed by agitation at room temperature for 20 minutes. The solutionwas dialized through an acetate buffer (pH 4.4) containing 1M sodiumchloride, added with 60 mg of sucrose, and then added with 1 ml of acarbonate buffer (pH 9.2) containing 3 mg of purified goat complementcomponent C1q and also containing 1M sodium chloride and 10% of sucrose.After agitating for 2 hours, the mixture was further added with 0.1 mlof a 4 mg/ml solution of sodium borohydride, and then allowed to standat 4° C. for additional 2 hours. Thereafter, the admixture was subjectedto gel filtration through a Sephacryl S-300 (Trade Name of PharmaciaFine Chemicals Co.) column, and the fraction having both of theperoxidase activity and the C1q activity was collected.

(2) Test

Using a serum having a CF antibody titer of 16, the reactions of thethus obtained peroxidase-labelled complement component C1q with a herpessimplex virus CF antigen and with a normal cell antigen were inspectedby means of the solid phase enzyme immunoassay to obtain the results asset forth in the following Table 1. In Table 1, the results of thisComparative Example are shown while comparing with the results ofExample 3.

                  TABLE 1                                                         ______________________________________                                                   Color Development                                                                         Color Development                                                 of Herpes CF                                                                              of Normal                                                         Antigen Well                                                                              Antigen Well                                           ______________________________________                                        Enzyme-Labelled C1q                                                                        0.544         0.078                                              of Example 3                                                                  Enzyme Labelled C1q                                                                        0.328         0.281                                              of Comparative                                                                Example 1                                                                     ______________________________________                                    

It should be appreciated from the results shown in Table 1 that theenzyme-labelled complement component C1q prepared by the conventionalprocess reacts with the normal antigen inselectively or non-uniquely andand has a low or feeble capability of reacting with the herpes simplexvirus CF antigen selectively or uniquely; whereas the enzyme-labelledcomplement component C1q prepared by the process of the invention isconsiderably lowered in inselective or non-unique reaction with thenormal antigen to have a sufficiently high capability of reaction withthe herpes simplex virus CF antigen.

EXAMPLE 4 Toxin-Conjugated Complement Component C1q

(1) Preparation

4mg of purified ricin A chain was disolved in 1.2 ml of a phosphatebuffer (pH 7.0) containing 20% of dimethylformamide, and added with 30μl of a phosphate buffer containing 3% of4-(maleimidemethyl)cyclohexane-1-carboxylic acid succineimide ester(hereinafter referred to as CHM) to react at room temperature for anhour. Then, the solution containing the reaction product was passedthrough a Sephadex G-25 column to obtain 2.3 mg of a CHM-conjugatedricin which was dissolved in 1 ml of 0.1M phosphate buffer (pH 6.0) andthen added with 2.5 mg of the reduced complement component C1q preparedby the same process as in Example 3 and dissolved in 0.5 ml of aphosphate buffer to react with the latter by allowing to stand themixture at 4° C. for 22 hours. The reaction mixture was subjected to gelfiltration using Sephacryl S-200 (Trade Name of Pharmacia Fine ChemicalsCo.) to obtain 1.9 mg of a ricin-conjugated complement component C1q.

(2) Test

The T cell was refined from the BALB/c mouse spleen cell primed withDNP-KLH by passing the primed cell through a Nylon wool column. The Tcell fraction was put into a RPMI-1640-10%FCS culture medium containinganti-mouse Ly-2,3 antiserum and 2 μg/ml of the aforementionedricin-conjugated complement component C1q, and then allowed to standstationarily at 37° C. for an hour. Thereafter, the processed cell wasrinsed with a Hanks' balanced salt solution, cultivated in aRPMI-1640-10%FCS culture medium containing mouse Interleukin 2 for 7days, and the distribution of the recovered cell Ly antigen was checkedto find that the number of cells having the Ly-1 antigen on the surfacesthereof were increased as large as 1.6 times of those of a control whichhad not been processed with the anti-mouse Ly-2,3 antiserum and thericin-conjugated complement component C1q.

EXAMPLE 5 Dyestuff-Conjugated Complement Component C1q

(1) Preparation

10 mg of purified bovine serum albumin was dissolved in 1 ml of 0.5Mcarbonate buffer and added with 0.4 mg of fluorescein isothiocyanate(hereinafter referred to as FITC). After reacting the mixture for 7hours, the reaction mixture was subjected to gel filtration to obtainbovine serum albumin combined with FITC. 8.2 mg of the bovine serumalbumin combined with FITC was then dissolved in 0.4 ml of a 0.1M sodiumphosphate buffer (pH 7.0), and added with 50 μl of a 90 mg/ml solutionof CHM in dimethylformamide for reaction at 30° C. for an hour. Afterremoving the insoluble materials by centrifugal separation, the buffersolution was exchanged to 0.1M phosphate buffer (pH 6.0). 0.56 ml of thesolution was added with 0.5 ml of a 0.1M phosphate buffer (pH 6.0)containing 8 mg of the reduced complement component C1q prepared inExample 1 and 5 mM sodium ethylenediamine tetra-acetate, and theadmixture was maintained at 4° C. for 18 hours. After then, theadmixture was filtered through a gel filter of Sepharose 6B column toobtain 12 ml of an eluate fraction having a molecular weight rangingwithin 400,000 to 900,000 and containing a reaction product between thecomplement component C1q and the bovine serum albumin combined with theFITC.

(2) Test

Separately, the spleen cells were removed from the BDF₁ mouse and passedthrough a Nylon column to obtain T-cells. The thus obtained T-cells, arabbit antiserum against mouse brain associated T-cell antigen and theeluate fraction prepared through the process desribed in the precedingparagraph were mixed together to form a mixture having a concentrationof 150 times of the final concentration. After maintaining the mixtureon ice for an hour and rinsing sufficiently, the cell was floated on thesurface of a 50% glycerin-phosphate buffer solution and observed througha flourescent microscope. The result was that 93% of the cells emittedfluorescent light to reveal that almost all of the cells were theT-cells.

EXAMPLE 6 Complement-Component C1q Conjugated with Donor or Acceptor forElectron

(1) Preparation

Chlorophyllin a was dissolved in distilled water so that a 1 mg/mlsolution was formed, and the pH value of the solution was adjusted withhydrochloric acid to pH 7.5, followed by addition of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chloride andethylenediamine so that the resultant reaction mixture contained 0.1M ofthe former and 0.8M of the latter. The mixture was allowed to stand forreaction for 120 minutes. The reaction product was purified by the useof CM-Sephadex (Trade Name of Pharmacia Fine Chemicals Co.), and 450 μgof aminoethylated chlorophyllin a was dissolved in 0.5 ml of a 0.1Mphosphate buffer (pH 7.0) containing 40% of dimethylformamide. Thesolution of purified aminoethylated chlorophyllin a was mixed with 0.5ml of a 40% dimethylformamide solution containing 2% of CHM, andmaintained at 30° C. for an hour to react with CHM. The reaction mixturewas subjected to gel filtration using Biogel P-2 (Trade Name of Bio-RadLaboratories Inc.) to obtain CHM-chrolophyllin a. 12 ml of the reducedcomplement component C1q prepared in accordance with the process asdescribed in Example 3 and 100 μg of the CHM-chrolophyllin a were putinto 1.5 ml of a 0.1M phosphate buffer (pH 6.0), and allowed to standstationarily at 4° C. for 18 hous, followed by gel filtration at which afraction having a molecular weight ranging within 400,000 to 600,000 waspicked up, whereby 7.5 mg of chrolophyllin-labelled complement componentC1q was obtained.

(2) Test

Separately, a chemically modified antigen electrode was prepared bycoating an antigen protein against herpes simplex virus on an SnO₂ Nesaelectrode so that the antigen protein was combined with the electrodethrough a covalent bond. The electrode was immersed in a 25mM phosphatebuffer (pH 6.95) containing 50 mM hydroquinone, a 1/50 finalconcentration of a human blood serum (CF =32) including an anti-herpessimplex virus, and 10 μg of the chrolophyllin-labelled complementcomponent C1q, and irradiated by a white light while maintaining theelectrode potential at 0.1 Vs·SCE, whereupon generation of photocurrentswas observed. The quantum efficiencies of photocurrents were about 9%.

EXAMPLE 7 Magnetizable Substance-Conjugated Complement Component C1q

(1) Preparation

Polystyrene microbeads each having amino groups at the surface thereofand containing micro grains of magnetite were suspended in 1 ml of 0.1Mphosphate buffer (pH 7.0) containing 20% dimethylformamide, and addedwith 40 μl of 0.1M phosphate buffer containing 2.5% CHM, followed bymoderate agitation at 30° C. for 60 minutes for reaction. After rinsingthe beads, they were suspended again in 1 ml of 0.1M phosphate buffer(pH 6.0) and added with 0.5 ml of another phosphate buffer containing1.8 mg of the reduced complement component C1q prepared by the processas described in Example 3, and then the admixture was allowed to reactat 4° C. for 18 hours under moderate agitation. The beads were rinsedwith a Veronal buffer solution (pH 7.4) containing 0.1% of gelatin, thesolution being referred to as GVB hereinafter, and then stored in theGVB at 4° C.

(2) Test

Separately, the spleen cells of X5563 tumor-bearing C3H/He mouse werecultivated on a culture medium containing IL-2, and the once rinsedcells were again floated on the same culture medium and mixed with thebeads-conjugated complement component C1q prepared by the processdescribed in the preceding paragraph and an anti I-Jk antiserum,followed by stationary standing at 37° C. for 2 hours. After the lapseof the pre-set time, the cells were collected and then the collectedcells were again floated gently, and an intense magnetical force wasapplied from the exterior of the container to capture the cells havingthe I-Jk antigens at the surfaces thereof. The cytotoxic activity of thecell left in the culture medium against the X5563 tumor cell wasrecognized to be about 1.4 times higher than that of the cell cultivatedfor 5 days in a simple IL-2.

EXAMPLE 8 Measurement of Complement-Binding Antibody

On a 96 well microtiter plate absorbing a complement-fixed antigen ofthe herpes simplex virus added, respectively, were 5 μl of each ofsample sera inactivated to have complement-binding titers of less than 4and 16, and then 95 μl of the peroxidase-labelled complement componentC1q in Example 1 (diluted to 100 times volume with a gelatine-Veronalbuffer solution) was added, followed by stationary standing at roomtemperature for an hour. Thereafter, each well was rinsed three timeswith a phosphate buffer containing 0.05% of Tween-20 (a surface activeagent produced and sold by Nakarai Chemicals LTD.), and added with 100μl of an H₂ O₂ -ABTS solution [2,2'-adino-di-(3-ethyl-benzothiazolinesulfate) solution containing hydrogen peroxide], followed by standing atroom temperature for an hour to complete the reaction. After adding with100 μl of an enzymatic reaction terminating agent, the light absorbanceat 414 nm was measured to find that the absorbance of the serum havingthe complement-binding titer of less than 4 was 0.029 while that of theserum having the complement-binding titer of 16 was 0.579.

EXAMPLE 9 Measurement of Complement-binding Antibody

Bovine blood serum albumin was dissolved in a saline solution bufferedby phosphate to prepare a solution having a concentration of 20 μg/ml,which was poured in each well of a 96 well microtiter plate and thenmaintained at room temperature for 2 hours to be absorbed by each well.After removing free bovine blood serum albumin, 50 μl of anti-bovineserum albumin rabbit antiserum (stepwisely diluted by 800 to 6400 timeswith a gelatin-Veronal buffer) and 50 μl of the β-D-galactosidase-labelled complement component C1q synthesized in Example 2 were added,and the microtiter plate was allowed to stand at room temperature for 30minutes. After rinsing each well, 100 μl of o-nitrophenylβ-D-galactoside solution (in a phosphate buffer solution having a pHvalue of 7.3) was added, followed by standing at room temperature for 60minutes, and then 0.1 ml of a 0.1M sodium carbonate solution toterminate or cease the enzymatic reaction. The light absorbances ofrespective wells were measured at a wavelength of 420 nm to find thatthe light absorbances were gradually varied from 0.421 to 0.063depending on the change in dosed amounts of antiserum.

EXAMPLE 10 Measurement of Complement-binding Antibody

A purified antigen of herpes simplex virus was absorbed by 6.35 mm ofpolystyrne beads which were put into a small test tube, and addedsimultaneously with 0.1 ml of a 10 times diluted solution of a solutionof each of inactivated test sera (having the complement-binding titersof 16 and less than 4) in a gelatine-Veronal buffer and with 0.1 ml of asolution of the peroxidase-labelled complement component C1q prepared inExample 1 diluted with the same buffer. The admixture was then allowedto stand stationarily at room temperature for an hour. After rinsing thebeads, they were transferred to another small test tube, and added with0.3 ml of an o-phenylenediamine solution to react at room temperaturefor 45 minutes. The reaction was terminated by the addition of 2 ml of 1N hydrochloric acid, and the light absorbances of the samples at thewavelength of 490 nm were measured. The sample having thecomplement-binding titer of less than 4 had a light absorbance of 0.18,whereas the sample having the complement-binding titer of 16 had a lightabsorbance of 0.408.

EXAMPLE 11 Measurement of Antigen

A 96 well microtiter absorbing guinea pig anti-herpes simplex virusantibody (Fab) was supplied with 0.1 ml of uterus cervix swabs of apatient, and stationarily held at room temperature for 60 minutes. Afterrinsing the plate with the PBS (a 0.85% saline-containing phosphatebuffer having a pH value of 7.4) for three times, each well was addedwith 0.05ml of either one of the inactivated guinea pig anti-herpessimplex virus sera (having the complement-binding antibody titers of 16to 32) and also with 0.05ml of the peroxidase-labelled complementcomponent C1q prepared in Example 1. The plate was held stationarily atroom temperature for 60 minutes. Then, each well was rinsed with PBScontaining 0.05% of Tween-20 (a surface active agent produced and soldunder such Trade Name from Nakarai Chemicals LTD.), and added with 0.01ml of H₂ O₂ -ABTS [2,2'-adino-di(3-ethyl-benzothiazolin sulfate)containing hydrogen peroxide]solution to be held at room temperature foran hour for reaction. Thereafter, 0.05 ml of a 0.05% aqueous sodiumnitride which acted to terminate the enzymatic reaction, and then thelight absorbances of respective sample wells at a wavelength of 414 nmwere measured to find that the sample well filled with uterus cervixswabs of a patient who was negative against the herpes simplex virus hadan absorbance of 0.030 and that the sample wells filled with uteruscervix swabs of a patient who were positive against the herpes simplexvirus had absorbances of 0.113, 0.300, 0.550 and so on.

EXAMPLE 12 Measurement of Antigen

According to a conventional process, a lymphocyte fraction was preparedfrom the mouse spleen cell, followed by rinsing with the PBS, and thenthe concentration of the cell was adjusted to 1×10⁷ /ml. 0.1 ml of thethus prepared lymphocyte fraction, 0.05 ml of antimouse Thy-1,3alloserum, and 0.05 ml of the FITC-labelled complement component C1q ofExample 5 were mixed together and allowed to stand at room temperaturefor an hour. The cells were then rinsed thoroughly with the PBS andobserved through a fluorescent microscope. The result revealed that 37%of the cells were fluorescent.

EXAMPLE 13 Measurement of Antigen

The lymphocytes in a blood of a leukemia patient were suspended in 1 mlof a phosphate buffer to prepare a suspension containing 1×10⁸ /ml oflymphocytes, and the suspension was processed by an ultra-sonicator for2 minutes. The homogenate was then clarified by centrifugal separation,and the supernatant was added with 0.5ml of DNA Sepharose to reacttherewith at 37° C. for 60 minutes. The DNA Sepharose was bound with DNArelated enzymes, such as DNA polymerase and terminal deoxynucleotidyltransferase (TdT). Then, 0.1 ml of the peroxidase-labelled complementcomponent C1q and an inactivated rabbit anti-TdT serum, followed byreaction at 37° C. for 30 minutes. After rinsing thoroughly with PBS,the DNA Sepharose was recovered, to which 1 ml of a solution of H₂ O₂-ABTS, was added, and the admixture was reacted at 37° C. for 60minutes. Then, 1 ml of a 0.05% aqueous solution of sodium nitride actingas a terminator for the reaction, and the light absorbance of thesupernatant was measured at a wavelength of 414 nm. It could be judgedthat the sample having a light absorbance value of not more than 0.075showed that the patient was negative to TdT and that the sample having alight absorbance value of not less than 0.100 showed that the patientwas positive to TdT and suffered from acute leukemia.

EXAMPLE 14 Measurement of Neutralizing Antibody

Two sample sera having, respectively, neutralizing antibody titers of 32and 128 to the HSV (Herpes Simplex Virus) were diluted with a phosphatebuffer to have the volumes four times as large as the initial volumes,heated at 56° C. for 30 minutes to be inactivated, and then furtherdiluted with the same buffer to have egiht times volumes. 0.1 ml foreach of the thus inactivated and diluted sample sera was mixed with 0.7ml of the same buffer containing 4×10³ pfu/ml of HSV, and then kept at37° C. for 60 minutes to proceed the reaction.

Separately, Vero cells had been cultivated through the momolayer cultureon a microplate, onto which a mixture of the serum and the HSV was addedat a content of 50 μl/well, and then the virus was absorbed by holdingthe plate in a culture filled with 0.5% carbon dioxide and maintained at37° C. for 60 minutes, with the addition of a maintenance mediumfollowed by cultivation for additional 24 hours. Then, the cell wasfixed by the use of methanol containing 3% of hydrogen peroxide.

After fixing by the methanol containing 3% of hydrogen peroxide, asdescribed in the preceding paragraph, 50 μl for each of human serahaving complement-fixing titers against the HSV diluted by 50 times witha gelatine-Veronal buffer (pH 7.4), respectively, of 16 and less than 4was poured into individual wells, and then each well was added with 0.2μg/50 μl/well of the peroxidase-labelled goat complement component C1qprepared in Example 3 and dissolved in the same buffer. After allowingto stand the microplate at room temperature for 2 hours, each well wasrinsed with a phosphate buffer solution containing 0.05% of Tween 20 forthree times, and then added with 0.7 ml/well of a H₂ O₂ -ABTS solutionto develop coloring of each well which was subjected to light absorbancedetermination conducted at a wavelength of 414 nm. The results are shownin Table 2.

                  TABLE 2                                                         ______________________________________                                        Serum for Detection                                                           of Residing Virus                                                                          OD414              (1)-(2)                                       ______________________________________                                        Serum for    Well (1)     Well (2)                                            Determination of                                                                           Added with   Added with                                          Presence or Absence of                                                                     Guinea Pig   Normal                                              Neutralizing Antibody                                                                      Anti-HSV Serum                                                                             Guinea Pig                                                                    Serum                                               Control      0.618        0.095     0.523                                     (Well Not Added                                                               with Sample Serum)                                                            Sample Serum 0.504        0.092     0.412                                     Having Neutralizing                                                           Titer of 32                                                                   Sample Serum Having                                                                        0.117        0.101     0.016                                     Neutralizing                                                                  Titer of 128                                                                  ______________________________________                                    

EXAMPLE 15 Measurement of Neutralizing Antibody

An anti-HSV positive human blood serum having a neutralizing antibodytiter of 128 and a negative human blood serum were diluted by four timeswith a phosphate buffer, and inactivated, and then a serial dilutionseries diluted by 4 to 512 times was prepared each for the both sera ona microtiter plate provided with a number of wells each having a volumeof 0.1 ml/well. Each well was filled with 0.1ml of a buffer containing4×10³ pfu/ml of HSV. The following procedures were the same as inExample 14 to measure or determine the OD₄₁₄. The results are plotted inthe graph illustrated in FIG.1 wherein the abscissa indicates thedilution rate of each serum and the ordinate indicate the OD₄₁₄. Asshown, for the positive serum, the dilution rate giving the value aslarge as 1/2 of the maximum OD₄₁₄ corresponds to the neutralizingantibody titer of 128.

EXAMPLE 16 Measurement of Intracellular Substance or Microorganism

A specimen to be inspected was picked up from a defected portions of HSVinfected patient (pendedum or labia), and suspended in 1 ml of culturemedium solution containing an antibiotic. 0.1 ml of the suspension wasinoculated to two wells of a microplate in which Vero cells had beenpreliminarily cultivated, and further cultivated at 37° C. for 22 hours.

After the completion of 22 hour cultivation, the cultivated cells werefixed with 3% hydrogen peroxide-methanol, and 50 μl of a human serumdiluted by 25 times with a gelatine-Veronal buffer (pH 7.4), the serumhaving an anti-HSV complement binding titer (CF titer) of 32 or lessthan 4, was put into individual wells together with 50 μl of a solutionin the same buffer containing 90 ng of the peroxidase-labelledcomplement component C1q prepared in Example 3. After reacting at roomtemperature for 2 hours, each well was rinsed with a phosphate buffercontaining 0.05% of Tween 20 for three times, added with 0.1ml/well of ahydrogen peroxide-ABTS solution (pH 4) followed by standing for an hourto develop coloring, and then the reaction was terminated by theaddition of 0.1 ml of a 0.01% sodium azide. Thereafter, the lightabsorbance of the reaction product in each well was measured. The welladded with the human blood serum having a CF titer of 32 had a lightabsorbance of 0.263, whereas the well added with the serum having a CFtiter of less than 4 had a light absorbance of 0.089. From those result,it could be confirmed that the HSV virion was present in the specimeninspected.

EXAMPLE 17 Measurement of Intracellular Substance or Microorganism

With the aim to cloning a cell producing carcino embryonic antigen, thecell T3M-4 producing CEA from pancreas tumor was diluted to the limit(i.e. to 1 cell/well), and then cultivated on a 96 well microplate for16 days. After removing the culture medium solution, 0.1 ml oftripsin-sodium ethylenediamine tetra-acetate was put into each well tofloat the cells, and then two plates preliminarily filled with 0.2ml/well of a fresh culture medium solution were replicated so thatrepricas containing 20 μl/well of floating cells were prepared. Theculture medium solution in one of the repricas was thrown away, followedby fixation of the cells with 3%H₂ O₂ -methanol, and then added with 0.1ml of a 1/400 time diluted rabbit anti-CEA antiserum diluted with GVB(gelatine-Veronal buffer) and 110 ng/0.1ml/well of theperoxidase-labelled complement component C1q. After reacting at roomtemperature for 2 hours and rinsing, coloring of the well was developedby the addition of a solution of the substrate of ABTS [diammonium(2,2-azi)-di{3-ethylbenzothiazolin sulfonic acid}]. The OD₄₁₄ ofrespective wells ranged within 0.127 to 0.386. The cell in the wellshowing the maximum OD₄₁₄ was picked up from the other reprica, andsubjected to expansion.

EXAMPLE 18 Measurement of Intracellular Substance or Microorganism

100 μg (0.1 ml) of purified α-fetoprotein and 0.1 ml of Freund completeadjuvant were mixed together and dosed into the abdorminal cavity of a 7week age Blb/C mouse. After 28 days from the dosage of theaforementioned materials, 100 μg (0.3 ml) of of AFP (α-fetoprotein) wasadditionally dosed, and after 3 days of the dosage of the AFP, the renalcells were picked up and fused with NS-1 cells. The cells were spreadover a 96 well plate at a concentration or distribution density of 1×10⁵/ml. From the first day to the sixteenth day after fusing, selection bythe HAT medium (hypoxantine-thymidine-aminopterine medium) wasconducted, and the antibody activity of the supernatant of eachcultivated well was inspected on the seventeenth day to reveal thatprodcution of antibody was recognized at a rate of 64/948 wells andproduction of anti-AFP antibody was recognized in two wells. The cellsin respective wells were utilized as coated specimens while beingprocessed by 3%H₂ O₂ -methanol to be fixed, and then added with 20 μl ofa goat anti-mouse IgG (γ-chain selectivity) diluted by 200 times andalso with 18 ng/20 μl of the peroxidase-labelled complement componentC1q, followed by stational standing for 2 hours. After rinsingthoroughly, development of coloring of each cell was effected in adiaminobenzidine solution, and the number of cells producing the IgG wascounted to find that the ratio of positive cells were 62% and 91%,respectively.

EXAMPLE 19 Measurement without Solid Carrier

0.5 ml of diluted solution of normal guinea pig serum, 0.5 ml of HerpesSimplex virus and 1 ml of the peroxidase-labelled complement componentC1q solution prepared in Example 1, were charged into a small test tube,mixed together and reacted at 0° C. for 90 minutes. Alternatively, thesame procedures were repeated except in that 0.5 ml diluted solution ofguinea pig serum solution immunized three times with Herpes Simplexvirus was used in place of 0.5 ml of diluted solution of normal guineapig serum. Then, 1 ml of each of polyethyleneglycol solutions was addedto each of the reaction products so that ultimate concentration reached3%. After each of the resulting reaction products was allowed to standat room temperature, precipitates were removed by centrifugalseparation. Each of these precipitates was again dissolved in 2 ml of abuffer solution so as to be again processed with polyethyleneglycol.Finally, a 0.175% H₂ O₂₋₋₀.04 % ABTS solution (pH 4.0) was added to eachof the thus processed solutions to develop the color and the OD 414value was measured with a spectrophotometer. It was found that the OD414 value for the immunized guinea pig serum was 1.025 while that forthe normal serum was 0.092.

EXAMPLE 20 Conjugated Complement COmponents C1q

4 mg of the goat complement component C1q prepared in Example 3 wasdissolved in a tris buffer solution (pH 7.5) and the resulting solutionwas reduced with dithiothreitol. The buffer solution was then replacedby gel filtration by a 0.1M solium phosphate buffer solution (pH 6.0)containing 5 mM of ethylenediamine tetra-acetate (EDTA), and thesolution was then concentrated to 0.2 mM. The resulting solution wasthen admixed with 0.2 ml of the same buffer solution containing 0.1mg ofN,N'-oxydimethylenedimaleimide and the resulting admixture wasmaintained at 4° C. for 22 hours. The unreacted materials were removedby gel filtration to produce 910 μg of conjugated complement componentsC1q.

EXAMPLE 21 Antibody-conjugated Complement Component C1q

2mg of immunoglobulin G to be specifically bound with sheep blood cells(anti-SRBC-IgG) was reacted with 150 μg of4-maleimidemethyl)cyclohexane-1-carboxylic acid succineimide ester (CHM)in a phosphate buffer solution (pH 7.0). After gel filtration, thereaction product was concentrated to 0.2 ml. The resulting product wasthen reacted with 3 mg of the reduced complement component C1q dissolvedin 0.2 ml of a phosphate buffer solution containing 2mM ofethylenediamine tetra-acetate (EDTA)(pH 7.0). After gel filtration, 520μg of the complement component C1q conjugated with the anti-SRBC-IgG wasobtained.

Upon adding the complement component C1q together with the SRBC to aliquid containing a blood serum antigen (BSA) and an anti-BSA antibody,agglutination of SRBC occurred to a more or less degree depending on theamount of the BSA antigen.

Although he present invention has been described with reference to thespecific examples, it should be understood that various modificationsand variations can be easily made by those skilled in the art withoutdeparting from the spirit of the invention. Accordingly, the foregoingdisclosure should be interpreted as illustrative only and not to beinterpreted in a limiting sense. The present invention is limited onlyby the scope of the following claims.

What is claimed is:
 1. A complement component C1q wherein a substance isconjugated via a sulfur atom to at least one site of said component,said site not being involved in binding immunoglobulins,said substancebeing selected from the group consisting of signal emitting substancesand cell function regulating substances, said signal emitting substancebeing a member selected from the group consisting of enzymes, coenzymes,enzyme substrates, dyestuffs and pigments, magnetizable substances,donors and acceptors for electron transference, radioactive substances,metal compounds, metal compositions and agglutinating substances, saidcell function regulating substance being selected from the groupconsisting of antibiotics, growth factors, hormones, cell activationfactors, cell division factors, anticancer agents and toxins.
 2. Thecomplement component C1q according to claim 1, wherein said enzyme isselected from the group consisting of peroxidases, alkalinephosphatases, galactosidases and alcohol dehydrogenases.
 3. Thecomplement component C1q according to claim 1, wherein said coenzyme isselected from the group consisting of nicotinamide adenine dinucleotide,nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotideand flavin adenine dinucleotide phosphate.
 4. The complement componentC1q according to claim 1, wherein said enzyme substrate is selected fromthe group consisting of o-nitrophenyl-β-D-galactopyranoside and3-hydroxysteroid.
 5. The complement component C1q according to claim 1,wherein said dyestuff and pigments are selected from the groupconsisting of hemoglobin, methylene blue and fluorescein isothiocyanate.6. The complement component C1q according to claim 1, wherein saidmagnetizable susbstance is selected from the group consisting ofcarbonic iron, iron-containing microcapsules and complexes of iron withproteins.
 7. The complement component C1q according to claim 1, whereinsaid donor and acceptor for electron transference are chlorophyl.
 8. Thecomplement component C1q according to claim 1, wherein said radioactivesubstance is selected from the group consisting of ¹²⁴ I-labelledalbumin, p-chloro(²⁰³ Hg)mercuribenzoic acid, N-ethyl(2,3-¹⁴ C)maleimideand iode(1-¹⁴ C)acetamide.
 9. The complement component C1q according toclaim 1, wherein said metal compound and said metal composition areselected from the group consisting of gold colloid and iron-containingmicrobeads.
 10. The complement component C1q according to claim 1,wherein said antibiotic is selected from the group consisting ofamphotericin B and actinomycin D.
 11. The complement component C1qaccording to claim 1, wherein said growth factor is selected from thegroup consisting of selenium compounds, insulin, transferrin andepidermal growth factor.
 12. The complement component C1q according toclaim 1, wherein said hormone is corticosteroid.
 13. The complementcomponent C1q according to claim 1, wherein said cell activation factoris a macrophage activation factor.
 14. The complement component C1qaccording to claim 1, wherein said cell division factor is a B celldivision factor.
 15. The complement component C1q according to claim 1,wherein said anticancer agent is mitomycin C.
 16. The complementcomponent C1q according to claim 1, wherein said toxin is ricin of toxinof castor bean.
 17. The complement component C1q according to claim 1,wherein said agglutinating substance is selected from the groupconsisting of fine particles of latexes, fine particles of microbeads, acomplement component C1q, a combination of an antibody and an antigencorresponding to said antibody, a combination of lectin and a materialhaving saccharide structure corresponding to said lectin, a combinationof biotin and avidin, and a combination of protein and IgM.
 18. Aprocess for preparing a substance-conjugated complement component C1q,comprising the steps of:(a) adding a reducing agent to a complementcomponent C1q to cleave at least one S--S bond present at a site notinvolved in binding immunoglobulins thereby to obtain a reducedcomplement component C1q having at least one exposed --SH group; and (b)conjugating a substance to said complement component C1q via saidexposed --SH group.
 19. The process according to claim 18, wherein saidreducing agent is a sulfur-containing compound.
 20. The processaccording to claim 19, wherein said reducing agent is selected from thegroup consisting of mercaptoethylamine, dithiothreitol,2-mercaptoethanol, cysteine and glutathione.
 21. The process accordingto claim 18, wherein said reducing agent is allowed to act on thecomplement component C1q which is dissolved in a buffer solution forpermitting the complement component C1q to be present stably.
 22. Theprocess according to claim 18, wherein said buffer solution is selectedfrom the group consisting of tris buffered saline and phosphate bufferedsaline.
 23. The process according to claim 18, wherein said reducingagent is allowed to act on said complement component C1q at -2° C. to45° C. for 30 seconds to 24 hours.
 24. The process according to claim18, further comprising a step of removing excess reducing agent afterthe completion of said step (a).
 25. The process according to claim 18,wherein said substance is selected from the group consisting of signalemitting substances and cell function regulating substances.
 26. Theprocess according to claim 18, wherein said substance is conjugateddirectly to said exposed --SH group.
 27. The process according to claim18, wherein said substance is conjugated to said exposed --SH groupindirectly via a second susbstance having coupling function.
 28. Theprocess according to claim 18, wherein said second substance having saidcoupling function has a group selected from the group consisting ofmaleimide residue and --SH group, and also has a group for coupling saidsubstance.
 29. The process according to claim 18, wherein said substanceis peroxidase extracted from horseradish, and wherein said secondsubstance having said coupling function is N-hydroxysuccinimide ester ofmaleimide.
 30. The process according to claim 18, wherein said substanceis conjugated to said reduced complement component C1q in a buffersolution for both of said substance to be conjugated and said reducedcomplement component C1q.
 31. The process according to claim 18 whereinsaid step (b) is a step of conjugating said substance indirectly via asecond susbstance having coupling function and via said exposed --SHgroup to said complement component C1q, and wherein said step (b) iseffected in a common buffer for said reduced complement component C1q,said second substance having coupling function and said substance to beconjugated.